CN107868841A - For detecting complete molecular labeling and its application of tall and big goatweed chromosome arm - Google Patents

For detecting complete molecular labeling and its application of tall and big goatweed chromosome arm Download PDF

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CN107868841A
CN107868841A CN201711240973.2A CN201711240973A CN107868841A CN 107868841 A CN107868841 A CN 107868841A CN 201711240973 A CN201711240973 A CN 201711240973A CN 107868841 A CN107868841 A CN 107868841A
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sequence
primer
primer pair
chromosome
tall
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叶兴国
王坤杨
王轲
林志珊
杜丽璞
李仕金
王静
石磊
陈海强
马晓兰
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Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae

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Abstract

The invention discloses a kind of complete molecular labeling and its application for being used to detect tall and big goatweed chromosome arm.Present invention protection Specific primer pair, is made up of the single strand dna shown in the sequence 28 of sequence 1 of sequence table.The primer combination can be applied to the detection of tall and big goatweed chromosome arm.The method that the present inventor is combined by RNA seq and bioinformatic analysis, recycle the tall and big goatweed addition line of one group of common wheat further to confirm simultaneously, have developed a set of specific molecular marker for being used to identify tall and big goatweed chromosome arm and chromosome segment.The present invention will be highly useful for the tall and big goatweed chromosome segment needed for transfer wheat breeding.

Description

For detecting complete molecular labeling and its application of tall and big goatweed chromosome arm
Technical field
The present invention relates to biological technical field, and in particular to a kind of be used to detect tall and big goatweed chromosome arm complete point Son mark and its application.
Background technology
Tall and big goatweed (2n=2x=14, SlSl, Aegilops longissima L.) and it is the one of Tribe Triticeae Aegilops Individual diploid kind, it be mainly distributed on Middle East Egypt and Palestine seashore, in addition, the ground such as Jordan also have it is a small amount of Distribution, belongs to annual plant.On evolutionary relationship, the genome of tall and big goatweed is compared with the 1 B gene group of hexaploid wheat It is close.Tall and big goatweed for biotic (such as rust, powdery mildew, cyst roundworm, root-knot nematode, Mai Ying flies and greenbug) and Abiotic stress (such as arid, cold and high salt) possesses very extensive resistance, and contains the Fineness genes such as special glutenin, Therefore it is the Fineness gene source of wheat resistance breeding and quality breeding.For example, the Pm13 of the 3S chromosomes from tall and big goatweed Gene, extensive resistance of the wheat to powdery mildew (PM) is imparted, and important function has been played in wheat breeding and production；Pass through Application to Pm13, multiple wheat breeds with mildew-resistance are cultivated, and be applied to Wheat Production；In addition, in height Big goatweed 1Sl2 genes that coding hmw glutenin subunit (HMW-GS) be present are found on chromosome long arm (1Sx2.3 and 1Sy16), in tall and big goatweed 1SlFind coding low molecular weight glutenin subunit be present on the short arm of a chromosome (LMW-GS) gene (1SLMW-s).
At present, for disease-resistant, the degeneration-resistant and Fineness gene that is contained on each bar chromosome of tall and big goatweed to common small The importing of wheat, the main or progress in the form of chromosome translocation.But the research report so far to tall and big goatweed is also It is less, cloned particularly with the DNA fragment specific on the specific chromosome of tall and big goatweed, molecular markers development it is also fresh Have been reported that.Therefore, the result of study on tall and big goatweed acquired at present, far can not also meet in tall and big goatweed The needs of a large amount of favorable genes easy bit transitions into wheat.So develop each chromosome arm of tall and big goatweed and coloured differently The special molecular labeling of body section, for the gene such as disease-resistant, degeneration-resistant and high-quality present in tall and big goatweed is easy with chromosome Position mode efficiently imports common wheat, formulates common wheat-tall and big goatweed transposition based material, cultivates new variety of wheat, improvement The characters such as disease-resistant wheat, drought resisting, salt tolerant and quality, there is material time meaning.
The content of the invention
It is an object of the invention to provide a kind of complete molecular labeling for being used to detect tall and big goatweed chromosome arm and its answer With.
Present invention firstly provides Specific primer pair, is made up of 14 primer pairs；
Primer pair 1 is made up of primer 1SL-F and primer 1SL-R；
The primer 1SL-F is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) sequence 1 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 1 The DNA molecular of identical function；
The primer 1SL-R is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) sequence 2 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 2 The DNA molecular of identical function；
Primer pair 2 is made up of primer 1SS-F and primer 1SS-R；
The primer 1SS-F is following (a5) or (a6)：
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) sequence 3 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 3 The DNA molecular of identical function；
The primer 1SS-R is following (a7) or (a8)：
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) sequence 4 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4 The DNA molecular of identical function；
Primer pair 3 is made up of primer 2 SL-F and primer 2 SL-R；
The primer 2 SL-F is following (b1) or (b2)：
(b1) single strand dna shown in the sequence 5 of sequence table；
(b2) sequence 5 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5 The DNA molecular of identical function；
The primer 2 SL-R is following (b3) or (b4)：
(b3) single strand dna shown in the sequence 6 of sequence table；
(b4) sequence 6 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 6 The DNA molecular of identical function；
Primer pair 4 is made up of primer 2 SS-F and primer 2 SS-R；
The primer 2 SS-F is following (b5) or (b6)：
(b5) single strand dna shown in the sequence 7 of sequence table；
(b6) sequence 7 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 7 The DNA molecular of identical function；
The primer 2 SS-R is following (b7) or (b8)：
(b7) single strand dna shown in the sequence 8 of sequence table；
(b8) sequence 8 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 8 The DNA molecular of identical function；
Primer pair 5 is made up of primer 3SL-F and primer 3SL-R；
The primer 3SL-F is following (c1) or (c2)：
(c1) single strand dna shown in the sequence 9 of sequence table；
(c2) sequence 9 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 9 The DNA molecular of identical function；
The primer 3SL-R is following (c3) or (c4)：
(c3) single strand dna shown in the sequence 10 of sequence table；
(c4) sequence 10 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 10 There is the DNA molecular of identical function；
Primer pair 6 is made up of primer 3SS-F and primer 3SS-R；
The primer 3SS-F is following (c5) or (c6)：
(c5) single strand dna shown in the sequence 11 of sequence table；
(c6) sequence 11 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 11 There is the DNA molecular of identical function；
The primer 3SS-R is following (c7) or (c8)：
(c7) single strand dna shown in the sequence 12 of sequence table；
(c8) sequence 12 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 12 There is the DNA molecular of identical function；
Primer pair 7 is made up of primer 4SL-F and primer 4SL-R；
The primer 4SL-F is following (d1) or (d2)：
(d1) single strand dna shown in the sequence 13 of sequence table；
(d2) sequence 13 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 13 There is the DNA molecular of identical function；
The primer 4SL-R is following (d3) or (d4)：
(d3) single strand dna shown in the sequence 14 of sequence table；
(d4) sequence 14 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 14 There is the DNA molecular of identical function；
Primer pair 8 is made up of primer 4SS-F and primer 4SS-R；
The primer 4SS-F is following (d5) or (d6)：
(d5) single strand dna shown in the sequence 15 of sequence table；
(d6) sequence 15 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 15 There is the DNA molecular of identical function；
The primer 4SS-R is following (d7) or (d8)：
(d7) single strand dna shown in the sequence 16 of sequence table；
(d8) sequence 16 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 16 There is the DNA molecular of identical function；
Primer pair 9 is made up of primer 5SL-F and primer 5SL-R；
The primer 5SL-F is following (e1) or (e2)：
(e1) single strand dna shown in the sequence 17 of sequence table；
(e2) sequence 17 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 17 There is the DNA molecular of identical function；
The primer 5SL-R is following (e3) or (e4)：
(e3) single strand dna shown in the sequence 18 of sequence table；
(e4) sequence 18 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 18 There is the DNA molecular of identical function；
Primer pair 10 is made up of primer 5SS-F and primer 5SS-R；
The primer 5SS-F is following (e5) or (e6)：
(e5) single strand dna shown in the sequence 19 of sequence table；
(e6) sequence 19 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 19 There is the DNA molecular of identical function；
The primer 5SS-R is following (e7) or (e8)：
(e7) single strand dna shown in the sequence 20 of sequence table；
(e8) sequence 20 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 20 There is the DNA molecular of identical function；
Primer pair 11 is made up of primer 6SL-F and primer 6SL-R；
The primer 6SL-F is following (f1) or (f2)：
(f1) single strand dna shown in the sequence 21 of sequence table；
(f2) sequence 21 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 21 There is the DNA molecular of identical function；
The primer 6SL-R is following (f3) or (f4)：
(f3) single strand dna shown in the sequence 22 of sequence table；
(f4) sequence 22 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 22 There is the DNA molecular of identical function；
Primer pair 12 is made up of primer 6SS-F and primer 6SS-R；
The primer 6SS-F is following (f5) or (f6)：
(f5) single strand dna shown in the sequence 23 of sequence table；
(f6) sequence 23 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 23 There is the DNA molecular of identical function；
The primer 6SS-R is following (f7) or (f8)：
(f7) single strand dna shown in the sequence 24 of sequence table；
(f8) have by sequence 24 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 24 There is the DNA molecular of identical function；
Primer pair 13 is made up of primer 7SL-F and primer 7SL-R；
The primer 7SL-F is following (g1) or (g2)：
(g1) single strand dna shown in the sequence 25 of sequence table；
(g2) sequence 25 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 25 There is the DNA molecular of identical function；
The primer 7SL-R is following (g3) or (g4)：
(g3) single strand dna shown in the sequence 26 of sequence table；
(g4) sequence 26 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 26 There is the DNA molecular of identical function；
Primer pair 14 is made up of primer 7SS-F and primer 7SS-R；
The primer 7SS-F is following (g5) or (g6)：
(g5) single strand dna shown in the sequence 27 of sequence table；
(g6) sequence 27 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 27 There is the DNA molecular of identical function；
The primer 7SS-R is following (g7) or (g8)：
(g7) single strand dna shown in the sequence 28 of sequence table；
(g8) sequence 28 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 28 There is the DNA molecular of identical function.
The purposes of the Specific primer pair is following (h1) or (h2) or (h3) or (h4)：
(h1) identification treats whether contain tall and big goatweed chromosome arm in measuring plants；
(h2) prepare for identifying the kit for treating whether contain tall and big goatweed chromosome arm in measuring plants；
(h3) identify and treat whether measuring plants are tall and big goatweed addition line；
(h4) prepare for plant identification whether be tall and big goatweed addition line kit.
The present invention also protects the application of the Specific primer pair, for following (h1) or (h2) or (h3) or (h4)：
(h1) identification treats whether contain tall and big goatweed chromosome arm in measuring plants；
(h2) prepare for identifying the kit for treating whether contain tall and big goatweed chromosome arm in measuring plants；
(h3) identify and treat whether measuring plants are tall and big goatweed addition line；
(h4) prepare for plant identification whether be tall and big goatweed addition line kit.
The present invention also kit of the protection containing the Specific primer pair；The purposes of the kit is identification plant to be measured Whether contain tall and big goatweed chromosome in thing or identify and treat whether measuring plants are tall and big goatweed addition line.
The present invention also protects the preparation method of the kit, including the step of each bar primer is individually packed.
The present invention also protects a kind of method identified and treat whether contain tall and big goatweed chromosome arm in measuring plants, including such as Lower step：
Using the STb gene for treating measuring plants as template, each primer pair being respectively adopted in the Specific primer pair enters performing PCR Amplification, makes the following judgment：
During using the primer pair 1：If obtaining 275bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 1S1Chromosome long arm；If 275bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 1S1Chromosome long arm；
During using the primer pair 2：If obtaining 752bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 1S1The short arm of a chromosome；If 752bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 1S1The short arm of a chromosome；
During using the primer pair 3：If obtaining 603bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 2S1Chromosome long arm；If 603bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 2S1Chromosome long arm；
During using the primer pair 4：If obtaining 862bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 2S1The short arm of a chromosome；If 862bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 2S1The short arm of a chromosome；
During using the primer pair 5：If obtaining 628bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 3S1Chromosome long arm；If 628bp amplified production can not be obtained, treat that measuring plants do not contain or candidate does not contain Tall and big goatweed 3S1Chromosome long arm；
During using the primer pair 6：If obtaining 935bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 3S1The short arm of a chromosome；If 935bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 3S1The short arm of a chromosome；
During using the primer pair 7：If obtaining 187bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 4S1Chromosome long arm；If 187bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 4S1Chromosome long arm；
During using the primer pair 8：If obtaining 209bp amplified production, treat measuring plants contain or candidate contain it is tall and big Goatweed 4S1The short arm of a chromosome；If 209bp amplified production can not be obtained, treat that measuring plants do not contain or candidate does not contain height Big goatweed 4S1The short arm of a chromosome；
During using the primer pair 9：If obtaining 645bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 5S1Chromosome long arm；If 645bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 5S1Chromosome long arm；
During using the primer pair 10：If obtaining 669bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 5S1The short arm of a chromosome；If 669bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 5S1The short arm of a chromosome；
During using the primer pair 11：If obtaining 150bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 6S1Chromosome long arm；If 150bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 6S1Chromosome long arm；
During using the primer pair 12：If obtaining 970bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 6S1The short arm of a chromosome；If 970bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 6S1The short arm of a chromosome；
During using the primer pair 13：If obtaining 782bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 7S1Chromosome long arm；If 782bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 7S1Chromosome long arm；
During using the primer pair 14：If obtaining 974bp amplified production, treat to contain in measuring plants or candidate contains height Big goatweed 7S1The short arm of a chromosome；If 974bp amplified production can not be obtained, treat not containing in measuring plants or candidate is free of There is tall and big goatweed 7S1The short arm of a chromosome.
In methods described, the reaction system of the PCR amplifications is concretely：Template DNA 100ng, sense primer (10 μ mol·L-1) 0.5 μ L, anti-sense primer (10 μm of olL-1) 0.5 μ L, 2 × Taq MasterMix (Mg2+, dNTP) 7.5 μ L, add ddH2O to 15 μ L.
In methods described, the response procedures of PCR amplifications are concretely：95℃5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C 1min, 35 circulations；72℃8min.
The present invention also protects a kind of method identified and treat whether contain tall and big goatweed chromosome arm in measuring plants, including such as Lower step：
Detect in plant genomic DNA to be measured with the presence or absence of primer pair 1 described in the Specific primer pair to primer pair 14 Target sequence, make the following judgment：
If the target sequence of the primer pair 1 in the STb gene be present, treat to contain tall and big goatweed 1S in measuring plants1Dye Colour solid is long-armed；If the target sequence of the primer pair 1 is not present in the STb gene, treat not containing tall and big goatweed 1S in measuring plants1 Chromosome long arm；
If the target sequence of the primer pair 2 in the STb gene be present, treat to contain tall and big goatweed 1S in measuring plants1Dye Colour solid galianconism；If the target sequence of the primer pair 2 is not present in the STb gene, treat not containing tall and big goatweed 1S in measuring plants1 The short arm of a chromosome；
If the target sequence of the primer pair 3 in the STb gene be present, treat to contain tall and big goatweed 2S in measuring plants1Dye Colour solid is long-armed；If the target sequence of the primer pair 3 is not present in the STb gene, treat not containing tall and big goatweed 2S in measuring plants1 Chromosome long arm；
If the target sequence of the primer pair 4 in the STb gene be present, treat to contain tall and big goatweed 2S in measuring plants1Dye Colour solid galianconism；If the target sequence of the primer pair 4 is not present in the STb gene, treat not containing tall and big goatweed 2S in measuring plants1 The short arm of a chromosome；
If the target sequence of the primer pair 5 in the STb gene be present, treat to contain tall and big goatweed 3S in measuring plants1Dye Colour solid is long-armed；If the target sequence of the primer pair 5 is not present in the STb gene, treat not containing tall and big goatweed 3S in measuring plants1 Chromosome long arm；
If the target sequence of the primer pair 6 in the STb gene be present, treat to contain tall and big goatweed 3S in measuring plants1Dye Colour solid galianconism；If the target sequence of the primer pair 6 is not present in the STb gene, treat not containing tall and big goatweed 3S in measuring plants1 The short arm of a chromosome；
If the target sequence of the primer pair 7 in the STb gene be present, treat to contain tall and big goatweed 4S in measuring plants1Dye Colour solid is long-armed；If the target sequence of the primer pair 7 is not present in the STb gene, treat not containing tall and big goatweed 4S in measuring plants1 Chromosome long arm；
If the target sequence of the primer pair 8 in the STb gene be present, treat to contain tall and big goatweed 4S in measuring plants1Dye Colour solid galianconism；If the target sequence of the primer pair 8 is not present in the STb gene, treat not containing tall and big goatweed 4S in measuring plants1 The short arm of a chromosome；
If the target sequence of the primer pair 9 in the STb gene be present, treat to contain tall and big goatweed 5S in measuring plants1Dye Colour solid is long-armed；If the target sequence of the primer pair 9 is not present in the STb gene, treat not containing tall and big goatweed 5S in measuring plants1 Chromosome long arm；
If the STb gene has the target sequence of the primer pair 10, treat to contain tall and big goatweed 5S in measuring plants1Dyeing Body galianconism；If the target sequence of the primer pair 10 is not present in the STb gene, treat not containing tall and big goatweed 5S in measuring plants1 The short arm of a chromosome；
If the target sequence of the primer pair 11 in the STb gene be present, treat to contain tall and big goatweed 6S in measuring plants1Dye Colour solid is long-armed；If the target sequence of the primer pair 11 is not present in the STb gene, treat not containing tall and big goatweed in measuring plants 6S1Chromosome long arm；
If the target sequence of the primer pair 12 in the STb gene be present, treat to contain tall and big goatweed 6S in measuring plants1Dye Colour solid galianconism；If the target sequence of the primer pair 12 is not present in the STb gene, treat not containing tall and big goatweed in measuring plants 6S1The short arm of a chromosome；
If the target sequence of the primer pair 13 in the STb gene be present, treat to contain tall and big goatweed 7S in measuring plants1Dye Colour solid is long-armed；If the target sequence of the primer pair 13 is not present in the STb gene, treat not containing tall and big goatweed in measuring plants 7S1Chromosome long arm；
If the target sequence of the primer pair 14 in the STb gene be present, treat to contain tall and big goatweed 7S in measuring plants1Dye Colour solid galianconism；If the target sequence of the primer pair 14 is not present in the STb gene, treat not containing tall and big goatweed in measuring plants 7S1The short arm of a chromosome.
The present invention also protects the primer pair 1 or primer pair 2 or primer pair 3 or primer pair 4 or primer pair 5 or primer pair 6 Or primer pair 7 or primer pair 8 or primer pair 9 or primer pair 10 or primer pair 11 or primer pair 12 or primer pair 13 or primer pair 14。
The present invention also protects primer combination A or primer combination B or primer combination C or primer combination D or primer combination E or drawn Thing combines F or primer combination G.
The primer combination A is made up of the primer pair 1 and primer pair 2.
The primer combination B is made up of the primer pair 3 and primer pair 4.
The primer combination C is made up of the primer pair 5 and primer pair 6.
The primer combination D is made up of the primer pair 7 and primer pair 8.
The primer combination E is made up of the primer pair 9 and primer pair 10.
The primer combination F is made up of the primer pair 11 and primer pair 12.
The primer combination G is made up of the primer pair 13 and primer pair 14.
The present invention also protects the primer pair 1 identifying tall and big goatweed 1S1Application in chromosome long arm, or, it is described Primer pair 2 is identifying tall and big goatweed 1S1Application in the short arm of a chromosome, or, the primer pair 3 are identifying tall and big goatweed 2S1 Application in chromosome long arm, or, the primer pair 4 are identifying tall and big goatweed 2S1Application in the short arm of a chromosome, Huo,Suo State primer pair 5 and identify tall and big goatweed 3S1Application in chromosome long arm, or, the primer pair 6 are identifying tall and big goatweed 3S1Application in the short arm of a chromosome, or, the primer pair 7 are identifying tall and big goatweed 4S1Application in chromosome long arm, or, The primer pair 8 is identifying tall and big goatweed 4S1Application in the short arm of a chromosome, or, the primer pair 9 are identifying tall and big goat Careless 5S1Application in chromosome long arm, or, the primer pair 10 are identifying tall and big goatweed 5S1Application in the short arm of a chromosome, Or, the primer pair 11 is identifying tall and big goatweed 6S1Application in chromosome long arm, or, the primer pair 12 are high in identification Big goatweed 6S1Application in the short arm of a chromosome, or, the primer pair 13 are identifying tall and big goatweed 7S1In chromosome long arm Using, or, the primer pair 14 is in the tall and big goatweed 7S of identification1Application in the short arm of a chromosome.
The present invention also protects the primer combination A identifying tall and big goatweed 1S1Application in chromosome arm, or, it is described Primer combines B and is identifying tall and big goatweed 2S1Application in chromosome arm, or, primer combination C are identifying tall and big goatweed 3S1Application in chromosome arm, or, primer combination D are identifying tall and big goatweed 4S1Application in chromosome arm, Huo,Suo State primer combination E and identify tall and big goatweed 5S1Application in chromosome arm, or, primer combination F are identifying tall and big goat Careless 6S1Application in chromosome arm, or, primer combination G are identifying tall and big goatweed 7S1Application in chromosome arm.
Plant described in any of the above concretely wheat or tall and big goatweed or wheat-tall and big goatweed addition line.It is described Wheat concretely China spring.The tall and big goatweed concretely tall and big goatweed PI542196.The wheat-tall and big goat Careless addition line concretely China spring-tall and big goatweed addition line DA1S#3, China spring-tall and big goatweed addition line DA2S#3, China spring-tall and big goatweed addition line DA3S#2, China spring-tall and big goatweed addition line DA4S#3, China spring-tall and big goatweed Addition line DA5S#3, China spring-tall and big goatweed addition line DA6S#3 or China spring-tall and big goatweed addition line DA7S#3.
The method that the present inventor is combined by RNA-seq and bioinformatic analysis, while recycle one group Common wheat-tall and big goatweed addition line further confirms, have developed it is a set of be used to identifying tall and big goatweed chromosome arm and The specific molecular marker of chromosome segment.It is of the invention to be for shifting the tall and big goatweed chromosome segment needed for wheat breeding Highly useful.
Brief description of the drawings
Fig. 1 is the PCR the results of primer pair 1 in embodiment 3.
Fig. 2 is the PCR the results of primer pair 2 in embodiment 3.
Fig. 3 is the PCR the results of primer pair 3 in embodiment 3.
Fig. 4 is the PCR the results of primer pair 4 in embodiment 3.
Fig. 5 is the PCR the results of primer pair 5 in embodiment 3.
Fig. 6 is the PCR the results of primer pair 6 in embodiment 3.
Fig. 7 is the PCR the results of primer pair 7 in embodiment 3.
Fig. 8 is the PCR the results of primer pair 8 in embodiment 3.
Fig. 9 is the PCR the results of primer pair 9 in embodiment 3.
Figure 10 is the PCR the results of primer pair 10 in embodiment 3.
Figure 11 is the PCR the results of primer pair 11 in embodiment 3.
Figure 12 is the PCR the results of primer pair 12 in embodiment 3.
Figure 13 is the PCR the results of primer pair 13 in embodiment 3.
Figure 14 is the PCR the results of primer pair 14 in embodiment 3.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, it is respectively provided with 3 repetitions and tests, as a result make even Average.
Tall and big goatweed PI542196：Bibliography " Wang Shunli Aegilops correlation of attributes protein coding gene molecules gram Grand and Proteomic analysis .2012, Capital Normal University, thesis for the doctorate "；The public can be from crop section of the Chinese Academy of Agricultural Sciences Research institute is learned to obtain.
Wheat breed China spring：Country of Institute of Crop Science, Chinese Academy of Agricultural Science Germplasm Resources of Farm Crop storehouse.
China spring-tall and big goatweed addition line DA1S#3 (has 21 pairs of+1 pair tall and big goatweeds of wheat China spring chromosome 1S1 chromosomes)：Bibliography " Qi Shanshan, Bai Fuqiang, Xia Qing, Zhang Yudan, Zheng Yayue, Liu Jinyan, Liu Wen pavilion wheat relationship kinds Category addition is tolerant to low-phosphorus stress Characters Identification and Genetic food .2016,17 (4)：710-718”；The public can be therefrom Crop science research institute of Academy of Agricultural Sciences of state obtains.
China spring-tall and big goatweed addition line DA2S#3 (has 21 pairs of+1 pair tall and big goatweeds of wheat China spring chromosome 2S1 chromosomes)：Bibliography " Qi Shanshan, Bai Fuqiang, Xia Qing, Zhang Yudan, Zheng Yayue, Liu Jinyan, Liu Wen pavilion wheat relationship kinds Category addition is tolerant to low-phosphorus stress Characters Identification and Genetic food .2016,17 (4)：710-718”；The public can be therefrom Crop science research institute of Academy of Agricultural Sciences of state obtains.
China spring-tall and big goatweed addition line DA3S#2 (has 21 pairs of+1 pair tall and big goatweeds of wheat China spring chromosome 3S1 chromosomes)：Bibliography " Qi Shanshan, Bai Fuqiang, Xia Qing, Zhang Yudan, Zheng Yayue, Liu Jinyan, Liu Wen pavilion wheat relationship kinds Category addition is tolerant to low-phosphorus stress Characters Identification and Genetic food .2016,17 (4)：710-718”；The public can be therefrom Crop science research institute of Academy of Agricultural Sciences of state obtains.
China spring-tall and big goatweed addition line DA4S#3 (has 21 pairs of+1 pair tall and big goatweeds of wheat China spring chromosome 4S1 chromosomes)：Bibliography " Qi Shanshan, Bai Fuqiang, Xia Qing, Zhang Yudan, Zheng Yayue, Liu Jinyan, Liu Wen pavilion wheat relationship kinds Category addition is tolerant to low-phosphorus stress Characters Identification and Genetic food .2016,17 (4)：710-718”；The public can be therefrom Crop science research institute of Academy of Agricultural Sciences of state obtains.
China spring-tall and big goatweed addition line DA5S#3 (has 21 pairs of+1 pair tall and big goatweeds of wheat China spring chromosome 5S1 chromosomes)：Bibliography " Qi Shanshan, Bai Fuqiang, Xia Qing, Zhang Yudan, Zheng Yayue, Liu Jinyan, Liu Wen pavilion wheat relationship kinds Category addition is tolerant to low-phosphorus stress Characters Identification and Genetic food .2016,17 (4)：710-718”；The public can be therefrom Crop science research institute of Academy of Agricultural Sciences of state obtains.
China spring-tall and big goatweed addition line DA6S#3 (has 21 pairs of+1 pair tall and big goatweeds of wheat China spring chromosome 6S1 chromosomes)：Bibliography " Qi Shanshan, Bai Fuqiang, Xia Qing, Zhang Yudan, Zheng Yayue, Liu Jinyan, Liu Wen pavilion wheat relationship kinds Category addition is tolerant to low-phosphorus stress Characters Identification and Genetic food .2016,17 (4)：710-718”；The public can be therefrom Crop science research institute of Academy of Agricultural Sciences of state obtains.
China spring-tall and big goatweed addition line DA7S#3 (has 21 pairs of+1 pair tall and big goatweeds of wheat China spring chromosome 7S1 chromosomes)：Bibliography " Qi Shanshan, Bai Fuqiang, Xia Qing, Zhang Yudan, Zheng Yayue, Liu Jinyan, Liu Wen pavilion wheat relationship kinds Category addition is tolerant to low-phosphorus stress Characters Identification and Genetic food .2016,17 (4)：710-718”；The public can be therefrom Crop science research institute of Academy of Agricultural Sciences of state obtains.
The design of embodiment 1, the tall and big each chromosome arm primer set of goatweed of detection
Tall and big goatweed PI542196 seedling leaves total serum IgEs are extracted, by transcript profile sequencing and sequence analysis, comparison, are obtained Obtained some primers for being used to identify tall and big goatweed chromosome arm.By comparing the performances such as sensitivity, specificity, one is obtained The primer for detecting tall and big goatweed chromosome arm is covered to combine.It is as follows：
For detecting tall and big goatweed 1S1(the primer size of primer pair 1 of chromosome long arm：275bp)：
1SL-F (sequence 1 of sequence table)：5'-CCATACACCAGTACATCCATA-3'；
1SL-R (sequence 2 of sequence table)：5'-GTACATTACCATCTGACATAGG-3'；
For detecting tall and big goatweed 1S1(the primer size of primer pair 2 of the short arm of a chromosome：752bp)：
1SS-F (sequence 3 of sequence table)：5'-TACCACATCCATCACTCCT-3'；
1SS-R (sequence 4 of sequence table)：5'-ATTCTCCACACTCCAATCAA-3'；
For detecting tall and big goatweed 2S1(the primer size of primer pair 3 of chromosome long arm：603bp)：
2SL-F (sequence 5 of sequence table)：5'-TGGTATCTCACGCCTTCG-3'；
2SL-R (sequence 6 of sequence table)：5'-AAGACTTACATTTGGGAAC-3'；
For detecting tall and big goatweed 2S1(the primer size of primer pair 4 of the short arm of a chromosome：862bp)：
2SS-F (sequence 7 of sequence table)：5'-ATTAACAACCGCAGGCTTA-3'；
2SS-R (sequence 8 of sequence table)：5'-ACACTTACCACTTCAGACAA-3'；
For detecting tall and big goatweed 3S1(the primer size of primer pair 5 of chromosome long arm：628bp)：
3SL-F (sequence 9 of sequence table)：5'-GGAACTCTGGACATATACTCA-3'；
3SL-R (sequence 10 of sequence table)：5'-TTCTGCTGCTCCTTGTTG-3'；
For detecting tall and big goatweed 3S1(the primer size of primer pair 6 of the short arm of a chromosome：935bp)：
3SS-F (sequence 11 of sequence table)：5'-AACACTTGTGCCAATGCA-3'；
3SS-R (sequence 12 of sequence table)：5'-GAGCCACTAACCAGGAAA-3'；
For detecting tall and big goatweed 4S1(the primer size of primer pair 7 of chromosome long arm：187bp)：
4SL-F (sequence 13 of sequence table)：5'-ACGGTTCCTATGGTGCTG-3'；
4SL-R (sequence 14 of sequence table)：5'-GCACAGCCTTGTCAGATC-3'；
For detecting tall and big goatweed 4S1(the primer size of primer pair 8 of the short arm of a chromosome：209bp)：
4SS-F (sequence 15 of sequence table)：5'-TTCTCGCAGGAAGTCTTG-3'；
4SS-R (sequence 16 of sequence table)：5'-TCATCAGTAACCGTTCCG-3'；
For detecting tall and big goatweed 5S1(the primer size of primer pair 9 of chromosome long arm：645bp)：
5SL-F (sequence 17 of sequence table)：5'-GGGTTTATCTAAGGAGGT-3'；
5SL-R (sequence 18 of sequence table)：5'-AGTAGTTGTGCCTATGTTCT-3'；
For detecting tall and big goatweed 5S1(the primer size of primer pair 10 of the short arm of a chromosome：669bp)：
5SS-F (sequence 19 of sequence table)：5'-CGTAACACAATGAGGACATC-3'；
5SS-R (sequence 20 of sequence table)：5'-GTGCGTATCCACATATAACC-3'；
For detecting tall and big goatweed 6S1(the primer size of primer pair 11 of chromosome long arm：150bp)：
6SL-F (sequence 21 of sequence table)：5'-CATTCCTAACATCGATAACAG-3'；
6SL-R (sequence 22 of sequence table)：5'-TCTTGTGTTAGTAGTAGATCAGT-3'；
For detecting tall and big goatweed 6S1(the primer size of primer pair 12 of the short arm of a chromosome：970bp)：
6SS-F (sequence 23 of sequence table)：5'-TCACTTGCTCTGCTTCGTA-3'；
6SS-R (sequence 24 of sequence table)：5'-TCAGCACCGTGACCAATT-3'；
For detecting tall and big goatweed 7S1(the primer size of primer pair 13 of chromosome long arm：782bp)：
7SL-F (sequence 25 of sequence table)：5'-GCGGTATGATTCACAGTTG-3'；
7SL-R (sequence 26 of sequence table)：5'-CCATATCTTATTGCCTGCTT-3'；
For detecting tall and big goatweed 7S1(the primer size of primer pair 14 of the short arm of a chromosome：974bp)：
7SS-F (sequence 27 of sequence table)：5'-GCTTAGAATCGCCTGGAG-3'；
7SS-R (sequence 28 of sequence table)：5'-AATGTGAGGTCGTTATCGTA-3'.
The foundation of each chromosome arm detection method of embodiment 2, tall and big goatweed
1st, plant genomic DNA to be measured is extracted.
2nd, using the STb gene that step 1 obtains as template, primer pair 1 to the primer pair 14 being respectively adopted in embodiment 1 is carried out PCR is expanded, and obtains pcr amplification product.
The reaction system of PCR amplifications：Template DNA 100ng, the μ L of sense primer 0.5, anti-sense primer 0.5 μ L, 2 × TaqMasterMix(Mg2+, dNTP) 7.5 μ L, add ddH2O to 15 μ L.
The form of primer solution has been added in reaction system for the sense primer and anti-sense primer, and every primer is in primer Concentration in solution is 10 μm of olL-1。
The response procedures of PCR amplifications：95℃5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 1min, 35 circulations；72℃ 8min, then 4 DEG C of preservations.
3rd, the pcr amplification product for obtaining step 2 enters row agarose gel electrophoresis detection, and electrophoresis result is analyzed, Analysis method is as follows：
Enter performing PCR amplification to template using primer pair 1, if obtaining 275bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 1S1Chromosome long arm；If 275bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 1S1Chromosome long arm；
Enter performing PCR amplification to template using primer pair 2, if obtaining 752bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 1S1The short arm of a chromosome；If 752bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 1S1The short arm of a chromosome；
Enter performing PCR amplification to template using primer pair 3, if obtaining 603bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 2S1Chromosome long arm；If 603bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 2S1Chromosome long arm；
Enter performing PCR amplification to template using primer pair 4, if obtaining 862bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 2S1The short arm of a chromosome；If 862bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 2S1The short arm of a chromosome；
Enter performing PCR amplification to template using primer pair 5, if obtaining 628bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 3S1Chromosome long arm；If 628bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 3S1Chromosome long arm；
Enter performing PCR amplification to template using primer pair 6, if obtaining 935bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 3S1The short arm of a chromosome；If 935bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 3S1The short arm of a chromosome；
Enter performing PCR amplification to template using primer pair 7, if obtaining 187bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 4S1Chromosome long arm；If 187bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 4S1Chromosome long arm；
Enter performing PCR amplification to template using primer pair 8, if obtaining 209bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 4S1The short arm of a chromosome；If 209bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 4S1The short arm of a chromosome；
Enter performing PCR amplification to template using primer pair 9, if obtaining 645bp amplified production, treat that measuring plants contain or waited Choosing contains tall and big goatweed 5S1Chromosome long arm；If 645bp amplified production can not be obtained, treat that measuring plants are not contained or waited Choosing does not contain tall and big goatweed 5S1Chromosome long arm；
Template is entered using primer pair 10 performing PCR amplification, if obtaining 669bp amplified production, treat measuring plants contain or Candidate contains tall and big goatweed 5S1The short arm of a chromosome；If 669bp amplified production can not be obtained, treat measuring plants do not contain or Candidate does not contain tall and big goatweed 5S1The short arm of a chromosome；
Template is entered using primer pair 11 performing PCR amplification, if obtaining 150bp amplified production, treat measuring plants contain or Candidate contains tall and big goatweed 6S1Chromosome long arm；If 150bp amplified production can not be obtained, treat measuring plants do not contain or Candidate does not contain tall and big goatweed 6S1Chromosome long arm；
Template is entered using primer pair 12 performing PCR amplification, if obtaining 970bp amplified production, treat measuring plants contain or Candidate contains tall and big goatweed 6S1The short arm of a chromosome；If 970bp amplified production can not be obtained, treat measuring plants do not contain or Candidate does not contain tall and big goatweed 6S1The short arm of a chromosome；
Template is entered using primer pair 13 performing PCR amplification, if obtaining 782bp amplified production, treat measuring plants contain or Candidate contains tall and big goatweed 7S1Chromosome long arm；If 782bp amplified production can not be obtained, treat measuring plants do not contain or Candidate does not contain tall and big goatweed 7S1Chromosome long arm；
Template is entered using primer pair 14 performing PCR amplification, if obtaining 974bp amplified production, treat measuring plants contain or Candidate contains tall and big goatweed 7S1The short arm of a chromosome；If 974bp amplified production can not be obtained, treat measuring plants do not contain or Candidate does not contain tall and big goatweed 7S1The short arm of a chromosome.
Embodiment 3, detection method checking
Detected materials：Tall and big goatweed PI542196, wheat breed China spring, China spring-tall and big goatweed addition line DA1S#3, China spring-tall and big goatweed addition line DA2S#3, China spring-tall and big goatweed addition line DA3S#2, China spring-height Big goatweed addition line DA4S#3, China spring-tall and big goatweed addition line DA5S#3, China spring-tall and big goatweed addition line DA6S#3 and China spring-tall and big goatweed addition line DA7S#3.
Extract the STb gene of detected materials seedling leaves；Using STb gene as template, examined using the method in embodiment 2 Survey.
As a result as represented in figures 1 through 14.In Fig. 1-14, swimming lane M is Marker 2000+, and swimming lane 2 is China spring-tall and big goat Careless addition line DA1S#3, swimming lane 3 are China spring-tall and big goatweed addition line DA2S#3, and swimming lane 3 is China spring-tall and big goatweed Addition line DA3S#2, swimming lane 4 are China spring-tall and big goatweed addition line DA4S#3, and swimming lane 5 is that China spring-tall and big goatweed is attached It is DA5S#3 to add, and swimming lane 6 is China spring-tall and big goatweed addition line DA6S#3, and swimming lane 7 is that China spring-tall and big goatweed adds It is DA7S#3, swimming lane 8 is wheat breed China spring, and swimming lane 9 is tall and big goatweed PI542196.
As a result show, the template DNA of detected materials is expanded using primer pair 1, only China spring-tall and big goatweed Addition line DA1S#3 and tall and big goatweed PI542196 obtain 275bp amplified production, do not have any amplification in other 7 materials Product；The template DNA of detected materials is expanded using primer pair 2, only China spring-tall and big goatweed addition line DA1S#3 752bp amplified production is obtained with tall and big goatweed PI542196, does not have any amplified production in other 7 materials；Using drawing Thing expands to the template DNA of 3 pairs of detected materials, only China spring-tall and big goatweed addition line DA2S#3 and tall and big goat Careless PI542196 obtains 603bp amplified production, does not have any amplified production in other 7 materials；Using primer pair 4 to be measured The template DNA of material is expanded, and only China spring-tall and big goatweed addition line DA2S#3 and tall and big goatweed PI542196 are obtained To 862bp amplified production, there is no any amplified production in other 7 materials；Template using primer pair 5 to detected materials DNA is expanded, and only China spring-tall and big goatweed addition line DA3S#2 and tall and big goatweed PI542196 obtain 628bp's Amplified production, there is no any amplified production in other 7 materials；The template DNA of detected materials is expanded using primer pair 6 Increasing, only China spring-tall and big goatweed addition line DA3S#2 and tall and big goatweed PI542196 obtain 935bp amplified production, There is no any amplified production in other 7 materials；The template DNA of detected materials is expanded using primer pair 7, it is only Chinese Spring-tall and big goatweed addition line DA4S#3 and tall and big goatweed PI542196 obtain 187bp amplified production, other 7 materials In there is no any amplified production；The template DNA of detected materials is expanded using primer pair 8, only China spring-tall and big goat Careless addition line DA4S#3 and tall and big goatweed PI542196 obtain 209bp amplified production, do not have any expansion in other 7 materials Increase production thing；The template DNA of detected materials is expanded using primer pair 9, only China spring-tall and big goatweed addition line DA5S#3 and tall and big goatweed PI542196 obtain 645bp amplified production, do not have any amplified production in other 7 materials； The template DNA of detected materials is expanded using primer pair 10, only China spring-tall and big goatweed addition line DA5S#3 and height Big goatweed PI542196 obtains 669bp amplified production, and 669bp amplified production is not obtained in other 7 materials；Using Primer pair 11 expands to the template DNA of detected materials, only China spring-tall and big goatweed addition line DA6S#3 and high mountain Sheep's hay PI542196 obtains 150bp amplified production, does not have any amplified production in other 7 materials；It is right using primer pair 12 The template DNA of detected materials is expanded, only China spring-tall and big goatweed addition line DA6S#3 and tall and big goatweed PI542196 obtains 970bp amplified production, does not have any amplified production in other 7 materials；Using primer pair 13 to be measured The template DNA of material is expanded, and only China spring-tall and big goatweed addition line DA7S#3 and tall and big goatweed PI542196 are obtained To 782bp amplified production, there is no any amplified production in other 7 materials；Template using primer pair 14 to detected materials DNA is expanded, and only China spring-tall and big goatweed addition line DA7S#3 and tall and big goatweed PI542196 obtain 974bp's Amplified production, 974bp amplified production is not obtained in other 7 materials.
It is above-mentioned test result indicates that, each tall and big goatweed dyeing of primer combination and detection method detection that the present invention establishes Body arm, there is specificity well.
<110>Institute of Crop Science, Chinese Academy of Agricultural Science
<120>For detecting complete molecular labeling and its application of tall and big goatweed chromosome arm
<160> 28
<210> 1
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<212> DNA
<213>Artificial sequence
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ccatacacca gtacatccat a 21
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<212> DNA
<213>Artificial sequence
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gtacattacc atctgacata gg 22
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<212> DNA
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taccacatcc atcactcct 19
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attctccaca ctccaatcaa 20
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tggtatctca cgccttcg 18
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aagacttaca tttgggaac 19
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attaacaacc gcaggctta 19
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acacttacca cttcagacaa 20
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ggaactctgg acatatactc a 21
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ttctgctgct ccttgttg 18
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aacacttgtg ccaatgca 18
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gagccactaa ccaggaaa 18
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acggttccta tggtgctg 18
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gcacagcctt gtcagatc 18
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ttctcgcagg aagtcttg 18
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<211> 18
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<220>
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tcatcagtaa ccgttccg 18
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<211> 18
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gggtttatct aaggaggt 18
<210> 18
<211> 20
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<400> 18
agtagttgtg cctatgttct 20
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cgtaacacaa tgaggacatc 20
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gtgcgtatcc acatataacc 20
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cattcctaac atcgataaca g 21
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tcttgtgtta gtagtagatc agt 23
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tcacttgctc tgcttcgta 19
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tcagcaccgt gaccaatt 18
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gcggtatgat tcacagttg 19
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ccatatctta ttgcctgctt 20
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gcttagaatc gcctggag 18
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aatgtgaggt cgttatcgta 20

Claims (10)

1. Specific primer pair, it is made up of 14 primer pairs；
Primer pair 1 is made up of primer 1SL-F and primer 1SL-R；
The primer 1SL-F is following (a1) or (a2)：
(a1) single strand dna shown in the sequence 1 of sequence table；
(a2) have by sequence 1 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 1 identical The DNA molecular of function；
The primer 1SL-R is following (a3) or (a4)：
(a3) single strand dna shown in the sequence 2 of sequence table；
(a4) have by sequence 2 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 2 identical The DNA molecular of function；
Primer pair 2 is made up of primer 1SS-F and primer 1SS-R；
The primer 1SS-F is following (a5) or (a6)：
(a5) single strand dna shown in the sequence 3 of sequence table；
(a6) have by sequence 3 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 3 identical The DNA molecular of function；
The primer 1SS-R is following (a7) or (a8)：
(a7) single strand dna shown in the sequence 4 of sequence table；
(a8) have by sequence 4 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4 identical The DNA molecular of function；
Primer pair 3 is made up of primer 2 SL-F and primer 2 SL-R；
The primer 2 SL-F is following (b1) or (b2)：
(b1) single strand dna shown in the sequence 5 of sequence table；
(b2) have by sequence 5 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 5 identical The DNA molecular of function；
The primer 2 SL-R is following (b3) or (b4)：
(b3) single strand dna shown in the sequence 6 of sequence table；
(b4) have by sequence 6 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 6 identical The DNA molecular of function；
Primer pair 4 is made up of primer 2 SS-F and primer 2 SS-R；
The primer 2 SS-F is following (b5) or (b6)：
(b5) single strand dna shown in the sequence 7 of sequence table；
(b6) have by sequence 7 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 7 identical The DNA molecular of function；
The primer 2 SS-R is following (b7) or (b8)：
(b7) single strand dna shown in the sequence 8 of sequence table；
(b8) have by sequence 8 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 8 identical The DNA molecular of function；
Primer pair 5 is made up of primer 3SL-F and primer 3SL-R；
The primer 3SL-F is following (c1) or (c2)：
(c1) single strand dna shown in the sequence 9 of sequence table；
(c2) have by sequence 9 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 9 identical The DNA molecular of function；
The primer 3SL-R is following (c3) or (c4)：
(c3) single strand dna shown in the sequence 10 of sequence table；
(c4) sequence 10 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 10 The DNA molecular of congenerous；
Primer pair 6 is made up of primer 3SS-F and primer 3SS-R；
The primer 3SS-F is following (c5) or (c6)：
(c5) single strand dna shown in the sequence 11 of sequence table；
(c6) sequence 11 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 11 The DNA molecular of congenerous；
The primer 3SS-R is following (c7) or (c8)：
(c7) single strand dna shown in the sequence 12 of sequence table；
(c8) sequence 12 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 12 The DNA molecular of congenerous；
Primer pair 7 is made up of primer 4SL-F and primer 4SL-R；
The primer 4SL-F is following (d1) or (d2)：
(d1) single strand dna shown in the sequence 13 of sequence table；
(d2) sequence 13 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 13 The DNA molecular of congenerous；
The primer 4SL-R is following (d3) or (d4)：
(d3) single strand dna shown in the sequence 14 of sequence table；
(d4) sequence 14 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 14 The DNA molecular of congenerous；
Primer pair 8 is made up of primer 4SS-F and primer 4SS-R；
The primer 4SS-F is following (d5) or (d6)：
(d5) single strand dna shown in the sequence 15 of sequence table；
(d6) sequence 15 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 15 The DNA molecular of congenerous；
The primer 4SS-R is following (d7) or (d8)：
(d7) single strand dna shown in the sequence 16 of sequence table；
(d8) sequence 16 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 16 The DNA molecular of congenerous；
Primer pair 9 is made up of primer 5SL-F and primer 5SL-R；
The primer 5SL-F is following (e1) or (e2)：
(e1) single strand dna shown in the sequence 17 of sequence table；
(e2) sequence 17 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 17 The DNA molecular of congenerous；
The primer 5SL-R is following (e3) or (e4)：
(e3) single strand dna shown in the sequence 18 of sequence table；
(e4) sequence 18 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 18 The DNA molecular of congenerous；
Primer pair 10 is made up of primer 5SS-F and primer 5SS-R；
The primer 5SS-F is following (e5) or (e6)：
(e5) single strand dna shown in the sequence 19 of sequence table；
(e6) sequence 19 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 19 The DNA molecular of congenerous；
The primer 5SS-R is following (e7) or (e8)：
(e7) single strand dna shown in the sequence 20 of sequence table；
(e8) sequence 20 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 20 The DNA molecular of congenerous；
Primer pair 11 is made up of primer 6SL-F and primer 6SL-R；
The primer 6SL-F is following (f1) or (f2)：
(f1) single strand dna shown in the sequence 21 of sequence table；
(f2) sequence 21 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 21 The DNA molecular of congenerous；
The primer 6SL-R is following (f3) or (f4)：
(f3) single strand dna shown in the sequence 22 of sequence table；
(f4) sequence 22 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 22 The DNA molecular of congenerous；
Primer pair 12 is made up of primer 6SS-F and primer 6SS-R；
The primer 6SS-F is following (f5) or (f6)：
(f5) single strand dna shown in the sequence 23 of sequence table；
(f6) sequence 23 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 23 The DNA molecular of congenerous；
The primer 6SS-R is following (f7) or (f8)：
(f7) single strand dna shown in the sequence 24 of sequence table；
(f8) sequence 24 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 24 The DNA molecular of congenerous；
Primer pair 13 is made up of primer 7SL-F and primer 7SL-R；
The primer 7SL-F is following (g1) or (g2)：
(g1) single strand dna shown in the sequence 25 of sequence table；
(g2) sequence 25 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 25 The DNA molecular of congenerous；
The primer 7SL-R is following (g3) or (g4)：
(g3) single strand dna shown in the sequence 26 of sequence table；
(g4) sequence 26 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 26 The DNA molecular of congenerous；
Primer pair 14 is made up of primer 7SS-F and primer 7SS-R；
The primer 7SS-F is following (g5) or (g6)：
(g5) single strand dna shown in the sequence 27 of sequence table；
(g6) sequence 27 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 27 The DNA molecular of congenerous；
The primer 7SS-R is following (g7) or (g8)：
(g7) single strand dna shown in the sequence 28 of sequence table；
(g8) sequence 28 by the substitution of one or several nucleotides and/or missing and/or addition and had into phase with sequence 28 The DNA molecular of congenerous.
2. the application of the Specific primer pair described in claim 1, for following (h1) or (h2) or (h3) or (h4)：
(h1) identification treats whether contain tall and big goatweed chromosome arm in measuring plants；
(h2) prepare for identifying the kit for treating whether contain tall and big goatweed chromosome arm in measuring plants；
(h3) identify and treat whether measuring plants are tall and big goatweed addition line；
(h4) prepare for plant identification whether be tall and big goatweed addition line kit.
3. the kit containing the Specific primer pair described in claim 1；The purposes of the kit is that measuring plants are treated in identification In whether containing tall and big goatweed chromosome or identify and treat whether measuring plants are tall and big goatweed addition line.
4. the preparation method of kit described in claim 3, including the step of each bar primer is individually packed.
5. a kind of identify the method for treating whether contain tall and big goatweed chromosome arm in measuring plants, comprise the following steps：
Using the STb gene for treating measuring plants as template, each primer pair being respectively adopted in the Specific primer pair enters performing PCR expansion Increase, make the following judgment：
During using the primer pair 1：If obtaining 275bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 1S1Chromosome long arm；If 275bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 1S1Chromosome long arm；
During using the primer pair 2：If obtaining 752bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 1S1The short arm of a chromosome；If 752bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 1S1The short arm of a chromosome；
During using the primer pair 3：If obtaining 603bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 2S1Chromosome long arm；If 603bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 2S1Chromosome long arm；
During using the primer pair 4：If obtaining 862bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 2S1The short arm of a chromosome；If 862bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 2S1The short arm of a chromosome；
During using the primer pair 5：If obtaining 628bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 3S1Chromosome long arm；If 628bp amplified production can not be obtained, treat measuring plants do not contain or candidate do not contain it is tall and big Goatweed 3S1Chromosome long arm；
During using the primer pair 6：If obtaining 935bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 3S1The short arm of a chromosome；If 935bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 3S1The short arm of a chromosome；
During using the primer pair 7：If obtaining 187bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 4S1Chromosome long arm；If 187bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 4S1Chromosome long arm；
During using the primer pair 8：If obtaining 209bp amplified production, treat that measuring plants contain or candidate contains tall and big goat Careless 4S1The short arm of a chromosome；If 209bp amplified production can not be obtained, treat that measuring plants do not contain or candidate does not contain high mountain Sheep's hay 4S1The short arm of a chromosome；
During using the primer pair 9：If obtaining 645bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 5S1Chromosome long arm；If 645bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 5S1Chromosome long arm；
During using the primer pair 10：If obtaining 669bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 5S1The short arm of a chromosome；If 669bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 5S1The short arm of a chromosome；
During using the primer pair 11：If obtaining 150bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 6S1Chromosome long arm；If 150bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 6S1Chromosome long arm；
During using the primer pair 12：If obtaining 970bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 6S1The short arm of a chromosome；If 970bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 6S1The short arm of a chromosome；
During using the primer pair 13：If obtaining 782bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 7S1Chromosome long arm；If 782bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 7S1Chromosome long arm；
During using the primer pair 14：If obtaining 974bp amplified production, treat to contain in measuring plants or candidate contains high mountain Sheep's hay 7S1The short arm of a chromosome；If 974bp amplified production can not be obtained, treat not containing in measuring plants or candidate does not contain height Big goatweed 7S1The short arm of a chromosome.
6. a kind of identify the method for treating whether contain tall and big goatweed chromosome arm in measuring plants, comprise the following steps：
Detect in plant genomic DNA to be measured with the presence or absence of primer pair 1 described in the Specific primer pair described in claim 1 to primer To 14 target sequence, make the following judgment：
If the target sequence of the primer pair 1 in the STb gene be present, treat to contain tall and big goatweed 1S in measuring plants1Chromosome is grown Arm；If the target sequence of the primer pair 1 is not present in the STb gene, treat not containing tall and big goatweed 1S in measuring plants1Chromosome It is long-armed；
If the target sequence of the primer pair 2 in the STb gene be present, treat to contain tall and big goatweed 1S in measuring plants1Chromosome is short Arm；If the target sequence of the primer pair 2 is not present in the STb gene, treat not containing tall and big goatweed 1S in measuring plants1Chromosome Galianconism；
If the target sequence of the primer pair 3 in the STb gene be present, treat to contain tall and big goatweed 2S in measuring plants1Chromosome is grown Arm；If the target sequence of the primer pair 3 is not present in the STb gene, treat not containing tall and big goatweed 2S in measuring plants1Chromosome It is long-armed；
If the target sequence of the primer pair 4 in the STb gene be present, treat to contain tall and big goatweed 2S in measuring plants1Chromosome is short Arm；If the target sequence of the primer pair 4 is not present in the STb gene, treat not containing tall and big goatweed 2S in measuring plants1Chromosome Galianconism；
If the target sequence of the primer pair 5 in the STb gene be present, treat to contain tall and big goatweed 3S in measuring plants1Chromosome is grown Arm；If the target sequence of the primer pair 5 is not present in the STb gene, treat not containing tall and big goatweed 3S in measuring plants1Chromosome It is long-armed；
If the target sequence of the primer pair 6 in the STb gene be present, treat to contain tall and big goatweed 3S in measuring plants1Chromosome is short Arm；If the target sequence of the primer pair 6 is not present in the STb gene, treat not containing tall and big goatweed 3S in measuring plants1Chromosome Galianconism；
If the target sequence of the primer pair 7 in the STb gene be present, treat to contain tall and big goatweed 4S in measuring plants1Chromosome is grown Arm；If the target sequence of the primer pair 7 is not present in the STb gene, treat not containing tall and big goatweed 4S in measuring plants1Chromosome It is long-armed；
If the target sequence of the primer pair 8 in the STb gene be present, treat to contain tall and big goatweed 4S in measuring plants1Chromosome is short Arm；If the target sequence of the primer pair 8 is not present in the STb gene, treat not containing tall and big goatweed 4S in measuring plants1Chromosome Galianconism；
If the target sequence of the primer pair 9 in the STb gene be present, treat to contain tall and big goatweed 5S in measuring plants1Chromosome is grown Arm；If the target sequence of the primer pair 9 is not present in the STb gene, treat not containing tall and big goatweed 5S in measuring plants1Chromosome It is long-armed；
If the STb gene has the target sequence of the primer pair 10, treat to contain tall and big goatweed 5S in measuring plants1Chromosome is short Arm；If the target sequence of the primer pair 10 is not present in the STb gene, treat not containing tall and big goatweed 5S in measuring plants1Dyeing Body galianconism；
If the target sequence of the primer pair 11 in the STb gene be present, treat to contain tall and big goatweed 6S in measuring plants1Chromosome It is long-armed；If the target sequence of the primer pair 11 is not present in the STb gene, treat not containing tall and big goatweed 6S in measuring plants1Dye Colour solid is long-armed；
If the target sequence of the primer pair 12 in the STb gene be present, treat to contain tall and big goatweed 6S in measuring plants1Chromosome Galianconism；If the target sequence of the primer pair 12 is not present in the STb gene, treat not containing tall and big goatweed 6S in measuring plants1Dye Colour solid galianconism；
If the target sequence of the primer pair 13 in the STb gene be present, treat to contain tall and big goatweed 7S in measuring plants1Chromosome It is long-armed；If the target sequence of the primer pair 13 is not present in the STb gene, treat not containing tall and big goatweed 7S in measuring plants1Dye Colour solid is long-armed；
If the target sequence of the primer pair 14 in the STb gene be present, treat to contain tall and big goatweed 7S in measuring plants1Chromosome Galianconism；If the target sequence of the primer pair 14 is not present in the STb gene, treat not containing tall and big goatweed 7S in measuring plants1Dye Colour solid galianconism.
7. primer pair 1 or primer pair 2 or primer pair 3 or primer pair 4 or primer pair 5 or primer pair 6 described in claim 1 or Primer pair 7 or primer pair 8 or primer pair 9 or primer pair 10 or primer pair 11 or primer pair 12 or primer pair 13 or primer pair 14.
8. primer combines A or primer combination B or primer combination C or primer combination D or primer combination E or primer combination F or primer Combine G；
The primer combination A is made up of the primer pair 1 described in claim 1 and primer pair 2；
The primer combination B is made up of the primer pair 3 described in claim 1 and primer pair 4；
The primer combination C is made up of the primer pair 5 described in claim 1 and primer pair 6；
The primer combination D is made up of the primer pair 7 described in claim 1 and primer pair 8；
The primer combination E is made up of the primer pair 9 described in claim 1 and primer pair 10；
The primer combination F is made up of the primer pair 11 described in claim 1 and primer pair 12；
The primer combination G is made up of the primer pair 13 described in claim 1 and primer pair 14.
9. the primer pair 1 described in claim 1 is identifying tall and big goatweed 1S1Application in chromosome long arm, or, right will The primer pair 2 described in 1 is asked to identify tall and big goatweed 1S1Application in the short arm of a chromosome, or, described in claim 1 Primer pair 3 is identifying tall and big goatweed 2S1Application in chromosome long arm, or, primer pair 4 described in claim 1 are being reflected Fixed tall and big goatweed 2S1Application in the short arm of a chromosome, or, primer pair 5 described in claim 1 are identifying tall and big goatweed 3S1Application in chromosome long arm, or, primer pair 6 described in claim 1 are identifying tall and big goatweed 3S1The short arm of a chromosome In application, or, primer pair 7 described in claim 1 identifying tall and big goatweed 4S1Application in chromosome long arm, or, Primer pair 8 described in claim 1 is identifying tall and big goatweed 4S1Application in the short arm of a chromosome, or, in claim 1 Described primer pair 9 is identifying tall and big goatweed 5S1Application in chromosome long arm, or, the primer pair described in claim 1 10 are identifying tall and big goatweed 5S1Application in the short arm of a chromosome, or, the primer pair 11 described in claim 1 are high in identification Big goatweed 6S1Application in chromosome long arm, or, primer pair 12 described in claim 1 are identifying tall and big goatweed 6S1 Application in the short arm of a chromosome, or, primer pair 13 described in claim 1 are identifying tall and big goatweed 7S1Chromosome long arm In application, or, primer pair 14 described in claim 1 identifying tall and big goatweed 7S1Application in the short arm of a chromosome.
10. the primer combination A described in claim 8 is identifying tall and big goatweed 1S1Application in chromosome arm, or, claim Primer combination B described in 8 is identifying tall and big goatweed 2S1Application in chromosome arm, or, the primer sets described in claim 8 Close C and identify tall and big goatweed 3S1Application in chromosome arm, or, the primer combination D described in claim 8 are tall and big in identification Goatweed 4S1Application in chromosome arm, or, primer combination E described in claim 8 are identifying tall and big goatweed 5S1Chromosome Application in arm, or, primer combination F described in claim 8 are identifying tall and big goatweed 6S1Application in chromosome arm, or, Primer combination G described in claim 8 is identifying tall and big goatweed 7S1Application in chromosome arm.
CN201711240973.2A 2017-11-30 2017-11-30 For detecting complete molecular labeling and its application of tall and big goatweed chromosome arm Pending CN107868841A (en)

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